Kit for assaying the cellular integrity of blood platelets

Abstract

There is provided a kit or reagent for assaying the cellular integrity (intactness) of blood platelets comprising means for determining the quantity of PKA released by a number of platelets into a certain volume of plasma. The assay is based on determining cAMP-dependent phosphorylation of vitronectin (protein S) in plasma using radioactively labelled [γ 32 P]ATP as phosphate donor. The assay can be based on the specific inhibition of the phosphorylation in blood fluid by protein kinase inhibitors. The invention further relates to pharmaceutical compositions for the treatment of pathological conditions associated with impaired platelet function in humans, which comprises administering to a person in need thereof a physiologically active quantity of a PKA inhibitor adapted to decrease or prevent vitronectin phosphorylation. The inhibitor can be synthetic or genetically engineered peptide.

What is claimed is: 
     
       1. A kit for assaying the cellular integrity of blood platelets which comprises a package, wherein said package contains PKA determining means for determining the quantity of PKA released by a number of platelets into a certain volume of plasma and wherein said PKA determining means comprises. (a) a tagged phosphate donor for use in determining cAMP-dependent phosphorylation of vitronectin; and   (b) heparin, heparin-sulfate or another compound which is related to heparin and binds to the heparin-binding domain of vitronectin.   
     
     
       2. A kit for assaying the cellular integrity of blood platelets which comprises a package, wherein said package contains PKA determining means for determining the quantity of PKA released by a number of platelets into a certain volume of plasma and wherein said PKA determining means comprise: (a) at least one member selected from the group consisting of anti-PKA, anti-phosphovitronectin and anti-vitronectin antibodies; and   (b) heparin, heparin-sulfate or another compound which is related to heparin and binds to the heparin-binding domain of vitronectin.   
     
     
       3. A kit for assaying the cellular integrity of blood platelets which comprises a package, wherein said package contains PKA determining means for determining the quantity of PKA released by a number of platelets into a certain volume of plasma and wherein said PKA determining means comprises: (a) a tagged phosphate donor for use in determining cAMP-dependent phosphorylation of vitronectin; and   (b) a physiological platelet agonist which stimulates the release of PKA.   
     
     
       4. A kit for assaying the cellular integrity of blood platelets which comprises a package, wherein said package contains PKA determining means for determining the quantity of PKA released by a number of platelets into a certain volume of plasma and wherein said PKA determining means comprises: (a) at least one member selected from the group consisting of anti-PKA, anti-phosphovitronectin and antivitronectin antibodies; and   (b) a physiological platelet agonist which stimulates the release PKA.   
     
     
       5. A kit in accordance with claim 1, wherein said PKA-determining means further comprises: (c) a physiological platelet agonist which stimulates the release of PKA.   
     
     
       6. A kit in accordance with claim 2, wherein said PKA-determining means further comprises: (c) a physiological platelet agonist which stimulates the release of PKA.   
     
     
       7. A kit for assaying the cellular integrity of blood platelets comprising a package containing: a container of a tagged phosphate donor capable of supplying phosphate in the cAMP-dependent phosphorylation of vitronectin;   a container in which a phosphorylation reaction in a blood plasma sample can take place; and   a container of heparin, heparin-sulfate or another compound which is related to heparin and binds to the heparin binding domain of vitronectin.   
     
     
       8. A kit for assaying the cellular integrity of blood platelets comprising a package containing: a container of a tagged phosphate donor capable of supplying phosphate in the cAMP-dependent phosphorylation of vitronectin;   a container in which a phosphorylation reaction in a blood plasma sample can take place; and   a container of a physiological platelet agonist which stimulates the release of PKA.   
     
     
       9. A kit in accordance with claim 7, wherein said tagged phosphate donor is ATP. 
     
     
       10. A kit in accordance with claim 8, wherein said tagged phosphate donor is ATP. 
     
     
       11. A kit for assaying the cellular integrity of blood platelets comprising a package containing: a container of a member selected from the group consisting of anti-IKA, anti-phosphovitronectin and antivitronectin antibodies;   a container in which a phosphorylation reaction in a blood plasma sample can take place; and   a container of heparin, heparin-sulfate or another compound which is related to heparin and binds to the heparin binding domain of vitronectin.   
     
     
       12. A kit for assaying the cellular integrity of blood platelets comprising a package containing: a container of a member selected from the group consisting of anti-PKA, anti-phosphovitronectin and antivitronectin antibodies;   a container in which a phosphorylation reaction in a blood plasma sample can take place; and   a container of a physiological platelet agonist which stimulates the release of PKA.   
     
     
       13. A kit in accordance with claim 1, further including instructions for use of the kit for assaying the cellular integrity of blood platelets. 
     
     
       14. A kit in accordance with claim 2, further including instructions for use of the kit for assaying the cellular integrity of blood platelets.